Approximately thirty percent of drugs in clinical use inhibit a disease-related enzymatic process (Copeland, R. A. Evaluation of enzyme inhibitors in drug discovery: a guide for medical chemists and pharmacologists (Wiley-Interscience, 2005)). Thus, the discovery of new enzyme inhibitors is an important area of research in biochemistry and pharmacology.
Polymerase inhibitors are valuable in both clinical and research settings. These inhibitors help in elucidating the mechanistic aspects of transcription and DNA replication, in mapping structure-function relationships, and in characterizing protein activity. Polymerase inhibitors are also among the most attractive drug targets. Knowledge about these inhibitors, their structures, and their mechanisms enable the design of new drugs such as anti-cancer agents, antiviral agents, and antibiotics that will be effective against new pathogens and antibiotic-resistant mutants of known pathogens. Because some of these inhibitors have been reported to induce and/or inhibit apoptosis, they also provide valuable tools for investigating apoptosis. Likewise, because some of these agents block specific steps of DNA transcription, polymerase inhibitors can help to elucidate the role of transcriptional control in regulating the expression of target genes in various healthy and disease states.
Drugs that target polymerase proteins involved in particular disease pathways are well known in the art. Reverse transcriptase inhibitors (RTIs), for example, are a class of antiretroviral drugs that target construction of viral DNA by inhibiting the activity of reverse transcriptase. There are two subtypes of RTIs with different mechanisms of action: nucleoside and nucleotide analogue RTIs are incorporated into the viral DNA leading to chain termination, while non-nucleoside-analogue RTIs act as competitive inhibitors of the reverse transcriptase enzyme. Current AIDs therapeutics that function by inhibiting HIV reverse transcriptase are described in the art (see, e.g., Bean et al., Appl Environ Microbiol 72:5670-5672 (2005)), and include Efavirenz (brand names SUSTIVA® and STOCRIN®) and Nevirapine (also marketed under the trade name VIRAMUNE®). Antibiotics that target polymerase proteins (e.g. rifampin) and cancer drugs that target polymerase proteins (e.g. cisplatin) and also known in the art.
Many drugs have been found to be efficacious in the treatment of cancer. These include diverse chemical compounds such as antimetabolites (e.g., methotrexate and fluorouracil), DNA-damaging agents (e.g., cyclophosphamide, cisplatin, and doxorubicin), mitotic inhibitors (e.g., vincristine), nucleotide analogues (e.g., 6-mercaptopurine), inhibitors of topoisomerases involved in DNA repair (e.g., etoposide), inhibitors of DNA polymerase (e.g., bleomycin), and intercalating agents like mitoxantrone.
Several drugs targeting enzymes of mammalian DNA replication are currently being investigated as promising candidates for cancer chemotherapy or as probes for understanding the roles of specific enzymes in DNA replication and repair. These potential drug candidates include corylifolin, bakuchiol, resveratrol, Neobavaisoflavone, and daidzein (see Sun et al., J. Nat. Prod. 61, 362-366 (1998)).
Other examples of DNA and RNA polymerase inhibitors include Actinomycin D, Streptomyces sp.: α-Amanitin, Amanita sp.; Aphidicolin, HSV replication inhibitor, BP5; Methyl α-Amanitin Oleate; Novobiocin, Sodium Salt; Rifampicin; RNA Polymerase III Inhibitor; and Actinomyin D, 7-Amino. Three polymerase inhibitors currently in Phase II trials for use against Hepatitis C Virus are Idenix/Novartis' valopicitabine (NM283); ViroPharma's HCV-796; and Roche's R1626. Roche/Idenix are also investigating valtorcitabine (val-LdC), a first strand viral DNA synthesis inhibitor in Phase II HCV trials after initial success as an HBV treatment.
DNA damaging agents provide some of the most successful treatments for cancer. The enzyme Poly(ADP-ribose)polymerase (i.e. PARP) can help repair DNA damage caused by the DNA damaging agents used to treat cancer. As PARP activity is often increased in cancer cells, it provides these cells with a survival mechanism. ABT-888 (Abott Oncology), for example, is an oral PARP-inhibitor developed by Abbott to prevent DNA repair in cancer cells and increase the effectiveness of common cancer therapies such as radiation and alkylating agents. Moreover, preclinical data indicates ABT-888 has improved the effectiveness of radiation and many types of chemotherapy in animal models of cancer.
These selected publications from the last 5 years illustrate the current state of the art with regard to the activity, mechanisms, and biochemistry of polymerase inhibitors:    Brown J A, Duym W W, Fowler J D, Suo, Z. (2007) “Single-turnover Kinetic Analysis of the Mutagenic Potential of 8-Oxo-7,8-dihydro-2′-deoxyguanosine during Gap-filling Synthesis Catalyzed by Human DNA Polymerases lambda and beta.” J Mol Biol. [Epub ahead of print]    Suo, Z., Abdullah M A. (2007) “Unique Composite Active Site of the Hepatitis C Virus NS2-3 Protease: A New Opportunity for Antiviral Drug Design.” ChemMedChem 2(3), 283-284,    Roettger M P, Fiala K A, Sompalli S, Dong Y, Suo Z. (2004) “Pre-steady-state kinetic studies of the fidelity of human DNA polymerase mu”, Biochemistry 43(43), 13827-38.    Fiala K A, Abdel-Gawad W, Suo Z. (2004) “Pre-steady-state kinetic studies of the fidelity and mechanism of polymerization catalyzed by truncated human DNA polymerase lambda”, Biochemistry 43(21), 6751-62.    Fiala, K. A & Suo Z.* (2004) Pre-Steady State Kinetic Studies of the Fidelity of Sulfolobus solfataricus P2 DNA Polymerase IV, Biochemistry 43, 2106-2115    Fiala, K. A & Suo Z.* (2004) Mechanism of DNA Polymerization Catalyzed by Sulfolobus solfataricus P2 DNA Polymerase IV, Biochemistry 43, 2116-2125    Fiala, K. A, Abdel-Gawad, W. & Suo Z.* (2004) Pre-Steady-State Kinetic Studies of the Fidelity and Mechanism of Polymerization Catalyzed by Truncated Human DNA Polymerase Lambda Biochemistry, accepted and in press.    Allison, A. J., Ray, A., Suo Z., Colacino, J. M., Andeson, K. S., Johnson, K. A. (2001) “Toxicity of Antiviral Nucleoside Analogs and the Human Mitochondrial DNA Polymerase”, J. Biol. Chem. 276, 40847-40857.
New drugs are the products of a long and involved drug development process, the first step of which is the discovery of compounds with promising activity. New enzyme inhibitors can be discovered by screening libraries of drug candidate compounds against a target enzyme. Conventional drug screening and validation approaches utilize micro- to milli-scale biochemical or cellular assays to detect downstream biochemical or cellular signatures of enzymatic interference. In view of the limitations of conventional drug screening methods, there remains a need in the art for improved methods and apparatuses for the detection of promising drug candidates.